Development of a Bone-Mimetic 3D Printed Ti6Al4V Scaffold to Enhance Osteoblast-Derived Extracellular Vesicles' Therapeutic Efficacy for Bone Regeneration.

Kenny Man,Mathieu Y Brunet,Maria Fernandez-Rhodes,David A Hoey,Soraya Williams,Sophie Louth,Angelica S Federici,Thomas E Robinson,Owen G Davies,Sophie C Cox

doi:10.3389/fbioe.2021.757220

Abstract

Extracellular Vesicles (EVs) are considered promising nanoscale therapeutics for bone regeneration. To date, EVs are typically procured from cells on 2D tissue culture plastic, an artificial environment that limits cell growth and does not replicate in situ biochemical or biophysical conditions. This study investigated the potential of 3D printed titanium scaffolds coated with hydroxyapatite to promote the therapeutic efficacy of osteoblast-derived EVs. Ti6Al4V titanium scaffolds with different pore sizes (500 and 1000 µm) and shapes (square and triangle) were fabricated by selective laser melting. A bone-mimetic nano-needle hydroxyapatite (nnHA) coating was then applied. EVs were procured from scaffold-cultured osteoblasts over 2 weeks and vesicle concentration was determined using the CD63 ELISA. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) following treatment with primed EVs was evaluated by assessing alkaline phosphatase activity, collagen production and calcium deposition. Triangle pore scaffolds significantly increased osteoblast mineralisation (1.5-fold) when compared to square architectures (P ≤ 0.001). Interestingly, EV yield was also significantly enhanced on these higher permeability structures (P ≤ 0.001), in particular (2.2-fold) for the larger pore structures (1000 µm). Furthermore osteoblast-derived EVs isolated from triangular pore scaffolds significantly increased hBMSCs mineralisation when compared to EVs acquired from square pore scaffolds (1.7-fold) and 2D culture (2.2-fold) (P ≤ 0.001). Coating with nnHA significantly improved osteoblast mineralisation (>2.6-fold) and EV production (4.5-fold) when compared to uncoated scaffolds (P ≤ 0.001). Together, these findings demonstrate the potential of harnessing bone-mimetic culture platforms to enhance the production of pro-regenerative EVs as an acellular tool for bone repair.

Highlights

Bone fractures caused by traumatic injury or common ageassociated disorders, such as osteoporosis, present an enormous healthcare and socioeconomic burden worldwide (Baroli, 2009; Dimitriou et al, 2011), with 10 million people suffering with musculoskeletal disorders in the United Kingdom alone (Chance-Larsen et al, 2019)
To visualise the scaffolds geometry, the CAD models (Figure 2A) and images from the produced scaffolds were obtained via 2D microscopy (Figure 2B), micro-CT scanning (Figure 2C) and Scanning Electron Microscopy (SEM) analysis (Figure 2D)
The findings showed a significantly elevated levels of intracellular calcium from scaffold-cultured cells when compared to the cells from 2D (>9.1-fold) (P ≤ 0.001), with the T500 (1.36-fold) and T1000 (1.27-fold) groups exhibited increased intracellular calcium when compared to the S500 and S1000 respectively (P ≤ 0.001) (Figure 4F)

Summary

Introduction

Bone fractures caused by traumatic injury or common ageassociated disorders, such as osteoporosis, present an enormous healthcare and socioeconomic burden worldwide (Baroli, 2009; Dimitriou et al, 2011), with 10 million people suffering with musculoskeletal disorders in the United Kingdom alone (Chance-Larsen et al, 2019). This is anticipated to increase further in the future, due to the growing ageing population and the demand for sustained quality of life in the older years (Amini et al, 2012). There is growing precedence to develop cell-free or acellular technologies to promote bone regeneration (Burdick et al, 2013; Wang et al, 2018)

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